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Molecular Genetics
Techniques
BIT 220
Chapter 20
What is Cloning?
Recombinant DNA technologies
1. Producing
Recombinant DNA
molecule
Incorporate gene of
interest into plasmid
(cloning vector)
2. Recombinant
molecule is cloned
(replicated)
Amplification of plasmid
by replication in host cell
Restriction
Endonucleases
TABLE 20.1
•enzymes that cuts double-stranded DNA
at RECOGNITION SEQUENCE
4 base cutters
6 base cutters
•Naming Protocol – after bacteria
•Staggered Cleavage vs. Blunt-end Cleavage
•Palindromes
•NOT species specific
•Need Ligase (T4 bacteriophage)
•forms phosphodiester linkage
FIGURE 20.1
Why use REs?
1)Cloning a gene into a
plasmid
2) Restriction Maps
Gene of interest can not have
restriction site within its
sequence
FIGURE 20.9
Plasmids
•Naturally found in bacteria
•extrachromosomal
•small circular DNA
•self-replicating each time bacteria divides
•double stranded
•can hold extra genes – YOUR gene of
interest
Cloning Vectors
FIGURE 20.2
Derived from naturally occurring plasmids or viruses
Scientists have engineered plasmids to carry these
characteristics
FEATURES
A. Selectable marker
B. Unique restriction site
Multiple cloning site ( polylinker)
FIGURE 20.3
C. Origin of replication
Plasmid Vector
1) small size (<10 kb for plasmid, also for
size of insert in can hold)
2) pBR322 FIGURE 20.4
Ampicillin resistance gene
Tetracycline resistance gene
4361 bp
origin of replication (these are specific to species)
high copy number
High-Copy Number Plasmids
10-100 copies per host cell
growth vectors
Low-Copy Number
1-4 copies per cell
expression vectors
Other vectors
1. Bacteriophage Vectors
FIGURE 20.5
Vector is 45 kb
Accommodates inserts 10-15 kb
2. Cosmids
combination of lambda phage and plasmid
hold inserts 35-45 kb
FIGURE 20.6
3. Artificial Chromosomes YAC (yeast artificial
Chromosomes - 500 kb inserts; BAC’s also
Shuttle vectors
Species use different regulatory sequences
transcription and translational
ori – can vary
promoters also different
Shuttle Vectors often have regulatory
elements for both prokaryotic and eukaryotic use
•created and amplified in E. coli
•expressed in mammalian cells
•Figure 20.7- example of use
Selection
for E coli which contain
plasmid
Insert at BamH1 site
disrupt ampR gene
Transform into E coli
Grow on agar that contains amp.
If colonies grow cells contain
plasmid
Types of Genes
A. Structural Genes transcribed and translated to
make enzymatic protein
B. Operator Genes control structural genes
C. Regulator Genes indirectly control operator
genes
OPERON
No Lactose Present
Lac Repressor protein
•made from regulatory gene (I)
•binds to operator
•RNA polymerase can NOT bind
•inhibits B-galactosidase transcription
Lactose Present
Lactose - Inducer molecule
•Lactose (IPTG) binds to lac repressor protein
•lac repressor protein can not bind to operator
•RNA polymerase binds to promoter
•B galactosidase is transcribed/translated
•X-gal is cleaved
•Cells turn BLUE
Transcriptional Control
pUC19
Amp R
lacI gene:
Repressor product
lac Z gene:
B-galactosidase
IPTG:
inducer of lac operon
A. Grow on
Ampicillin those with plasmid
(transformed cells) grow
IPTG
X gal
B. Unmodified plasmid - blue colonies
With insert - white colonies
insert disrupts lacZ gene
No Insert
IPTG induces the lac operon
Lac Z gene produces part of  gal
 -gal cleaves X gal
Colonies Turn Blue
WITH INSERT
Gene of interest inserted at MCS
Interrupts LacZ gene
B gal can NOT be made
X gal can NOT be cleaved
White Colonies
Genomic Library
Definition:
DNA clones which collectively contain
all of the genomic DNA of the source
organism
A. Genomic DNA library
B. cDNA library FIGURE 20.11
Procedure:
A. Cut entire genome with RE
B. Clone all fragments into vectors
C. Transform cells
Identification of Genes???
A. DNA hybridization
B. Immunological screening
antibody against protein
C. Gene Selection
Complementation Screening
HybridizationScreening Libraries
FIGURE 20-12
1. Plate bacteria on agar
2. Replica plate
3. Lyse cells
4. Denature double-stranded DNA
5. Transfer to filter (Nitrocellulose)
6. Incubate with a labeled probe
100-1000 bp
80% match over 50 base pairs
Where do we get probe?
DNA from related organism
Chemically Synthesize it from AA sequence
Chemical Synthesis of
DNA
Gene Machines OR DNA synthesizers
-automated chemical reactions which
synthesize single-stranded
oligonucleotides (50)
USES:
1) hybridization probes
2) primers for PCR
3) linkers for cloning
4) alter sequences of clones genes
mutagenesis
codon optimization
Sequencing of Nucleic
Acids
Sanger Method
enzymatic
dideoxynucleotide method
Maxam and Gilbert
chemical procedure
Sequencing protocol
DNA template to be sequenced
Primer - complementary sequence (17-24-mer)
to beginning of template
DNA polymerase
4 dNTPs
One radioactive dNTP
All tubes have all previously mentioned reactants
Did- dideoxynucleotide – missing other oxygen
In Tube 1: didATP
Tube 2 : didTTP
Tube 3 : didGTP
Tube 4 : didCTP
Autoradiograph
Read
bottom
to
top
Deduce
complement
strand
250-350 nt can be sequenced per autoradiograph
For very large pieces of DNA (5000 bp) use PRIMER WALKING
Primer Walking
Polymerase Chain
Reaction
Amplify a single piece of DNA to make rare
sequences abundant
Reactants
•
•
•
•
original piece of DNA (double stranded)
primer (second strand)
nucleotides
DNA polymerase (Taq)
•isolated from bacterium
•thermostable
PCR
Figure 20.24
Procedure:
1. Denature double stranded DNA with
high temp
95oC for 1 minute
2. Renature (Anneal)
Cool reaction 55oC
primers attach
3. Synthesis:
Raise temp to 75oC
complementary strands are
synthesized
4. Repeat Heat /Cooling Cycle
(each cycle 3-5 minutes)
Uses of PCR
1. Generate cDNA from mRNA
2. Detect mutations
3. To produce mutations
4. For DNA sequencing
5. Assemble whole genes from synthetic oligo
Blots
1. Electrophoreses
agarose, acrylamide (smaller)
2. Transfer FIGURE 20.19
nitrocellulose
nylon
3. Probe
A. Southern-DNA
B. Northern RNA
which genes are being expressed
C. Western-protein
FIGURE 20.22
Microarray Technology
RFLPs
• Restriction fragment length
polymorphisms
• Help find a change in the
sequence by adding/eliminating
a restriction site
• E.g., GAATTC –Glu/Phe also
site for Eco RI
• GAATAC – eliminates EcoR1
site and also now amino acids
are Glu/Tyr
• Can predict changes in sizes
expected when probed
• Do example on board
Double Digests of DNA
• Go over Figure 20.25 and
Problem 20.25, page 512.
• How to determine order of
restrictions sequences when
DNA is digested with one or
more restriction enzymes.